Serine protease (SP) is a kind of important and widely distributed proteolytic enzyme, which participates in a series of important physiological processes, including digestion, blood coagulation, embryonic development and immune response. But there are only a few preliminary reports about SP in crustaceans. Shrimp farming is constantly threatened by diseases. Further research on shrimp immune defense mechanism will provide clues for finding new ideas of disease resistance. In this study, the full-length cDNA sequence of a novel serine protease homologs (SPH) gene (Lv-SPH, GenBank accession number: KR020739) was cloned from hemocytes of Litopenaeus vannamei according to our previous transcriptome results. The sequence characteristics were analyzed by bioinformatics method. Tissue distribution and expression profile in response to virus infection were discussed by real-time fluorescence quantitative PCR. Bioinformatics analysis showed that the total length of the gene was 1 249 bp, the open reading frame (ORF ) region was 1 005 bp encoding 334 aa with 156 bp 5′-UTR and 88 bp 3′-UTR. The deduced amino acid sequence contained a signal peptide sequence with 16 amino acids at the amino end. SMART analysis showed that Lv-SPH contained a clip domain and a highly conserved SP domain (Tryp-SPc), which had two of the three active catalytic sites (His and Asp), and the third one is Gly. Lv-SPH was expressed in different tissues of L. vannamei. The main expression was in hemocyte, followed by hepatopancrease, heart and intestine. The lowest expression level was in muscle. Real time fluorescence quantitative PCR analysis showed that white spot syndrome virus (WSSV) could induce the up-regulated expression of Lv-SPH significantly. The relative expression of Lv-SPH increased significantly at 24 h and reached the highest level at 48 h, which was about three times that of the control group. Lv-SPH has typical characteristics of SPH family members and has tissue expression specificity. WSSV can significantly induce the up-regulated expression of Lv-SPH gene, indicating that it may be involved in the immune response process of L. vannamei induced by WSSV. The results provide a reference for further exploring shrimp innate immune regulation mechanism, and have potential application value in shrimp healthy culture and disease control.